Mansf. Kulturpfl. (A Valuable Medicinal Plant) from Shoot Bud Explants

Home , Boesenbergia rotunda, Curcuma amada, Curcuma zedoaria

Full Length Research Paper

Rapid micropropagation of Boesenbergia rotunda (L.) Mansf. Kulturpfl. (a valuable medicinal plant) from shoot bud explants

Nor Azma Yusuf 1,2 *, M. M. Suffian Annuar 1 and Norzulaani Khalid 1

1Biotechnology and Bioproduct Research Cluster, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2Faculty of Plantation and Agro biotech, Faculty of Applied Sciences, University Technology MARA (Uitm) 40450 Shah Alam, Selangor, Malaysia.

Accepted 7 December, 2010

A successful protocol was developed for mass propagation of Boesenbergia rotunda (L.) Mansf. Kulturpfl., an important medicinal plant. Numerous shoots were induced from young shoot bud of B. rotunda mature rhizome on Murashige and Skoog (1962) medium supplemented with 30.0 g/l sucrose, 2.0 g/l gelrite, different concentrations of 6-benzylaminopurine (BAP) and α-naphtaleneacetic acid (NAA). Plant medium supplemented with different concentrations of BAP alone or with NAA produced varying degree of multiple shoots. A supplementation of 2.0 mg/l BAP and 0.5 mg/l NAA gave the best result. Ninety percent of the explants induced multiple shoots within 10 to 14 days of inoculation with five maximum numbers of shoots per explant. The numbers of multiple shoots was low during initial subculture but increased after third subculture and were slightly decreased after fourth subculture. Rooting was spontaneous in almost all the treatments after 10 to 14 days of culture. Micropropagated plantlets were successfully acclimatized.

Key words: Boesenbergia rotunda , micropropagation, medicinal plant, Zingiberaceae.

INTRODUCTION

Boesenbergia rotunda (L) Mansf. Kulturpfl. (Larsen, 1989; Murakami et al., 1993; Tuchnida et al., 2002; 1996), is a perennial herbaceous plant that belongs to the Fahey and Stephenson, 2002; Tewtrakul et al., 2003). In family Zingiberaceae and is believed to have originated a recent study, 4-hydroxypanduratin A and panduratin A, from India and South-East Asia region. The pharma- isolated from the rhizome of B. rotunda were found to cological importance of this plant is mainly due to the show high inhibitory activity towards dengue-2 virus pro- presence of flavanoids, essential oil and chalcones tease at 120 ppm (Tan et al., 2006). (Jaipetch et al., 1982 ; Pandji et al., 1993; Trakoontivakorn B. rotunda is traditionally propagated by vegetative et al., 2001). Moreover, rhizomes contain a potential techniques using a rhizome segment which is lengthy for antipyretic, analgesic, anti-mutagenic, anti-inflammatory, large-scale multiplication. Moreover, many of the Zingibe- antioxidant enzymes and anti-human immunodeficiency raceae species are susceptible to rhizome and soft rot virus 1 (anti-HIV-1) protease inhibition (Pathong et al., diseases, leaf spot and easily infected with pathogens such as Coleotrichum species (Balachandran et al ., 1990; Chan and Thong, 2004). A rapid multiplication method is required to supply sufficient disease-free plant material for large scale cultivation using in vitro propa- *Corresponding author. E-mail: [email protected] or gation. In vitro propagation of Zingiberaceae has already [email protected]. Tel: +603 79674380. Fax: +603 79674178. been reported, for example Alpinia galanga (Inden and Abbreviations: BAP, N6-Benzylaminopurine; NAA, α- Asahir , 1988) and Zingiber officinale (Hosoki and Sagawa, naphthalene acetic acid; MS, Murashige and Skoog; HIV, 1977; Pillai and Kumar, 1982; Inden and Asahira, 1988; human immunodeficiency virus. Bhagyalakshmi and Singh, 1988; Ikeda and Tanabe Yusuf et al. 1195

1989). Several investigators reported micropropagation of Acclimatization ginger either from buds (Nadgauda et al., 1980) or shoot In vitro rooted plantlets of B. rotunda (Figure 1E) were removed tips (Inden and Asahira, 1988), meristems (Bhagyalakshmi from the solid MS medium and the roots were washed under and Singh, 1988), or even from callus (Malamug et al., running tap water to remove residual agar. Each plantlet was then 1991; Nirmal et al., 1992). In vitro clonal multiplication of planted into pots containing mixture of organic soil and sand (1:1) Curcuma species through rhizome buds also has been without direct sunlight. The percentage of surviving plantlets was reported (Nadgauda et al., 1978; Balachandran et al., recorded after four weeks of transfer. 1990; Barthakur and Bordoloi, 1992; Salvi et al., 2002; Loc et al., 2005, Yusuf et al., 2007; Naz et al., 2009). How- ever , to date, there are no reports on the rapid micropropa- RESULTS AND DISCUSSION gation of B. rotunda , which will be discussed in this paper. Initiation of shoot buds

MATERIALS AND METHODS Establishment of contamination free cultures was a major task due to the fact that the explants originated from Establishment of aseptic explants underground rhizomes (Hosoki and Sagawa, 1977). In this study, rhizomes were initially sprouted on soil free Mature rhizomes of B. rotunda were purchased from herbal supplier condition until shoot buds appearred. These shoot buds at a local Market in Kuala Lumpur, Malaysia. The rhizomes were cleaned and rinsed. The cleaned rhizomes were then placed in an were excised from rhizomes and were used as explants. open container to allow shoots to sprout two to four cm in length. The buds from the rhizomes of B. rotunda that were The sprouting buds were collected and washed with 20% (v/v) surface sterilized with mercury chloride solution and com- clorox for 15 min. Under aseptic conditions, shoots were surface mercial bleach (Clorox ) solution for 15 min could sterilized with 0.5% (w/v) aqueous solution of mercuric chloride establish more than 70% aseptic and surviving explants (HgCl 2) solution for five minutes and followed by three rinses in sterile distilled water. The shoot buds had their external leaves and remained free of contamination after four (4) weeks removed and trimmed down until the size ranged from 0.5 to 0.8 in MS medium. Chan and Thong (2004) also reported mm and used as explants (Figure 1A to C). They were then that the use of HgCl 2 with two-stage surface sterilization inoculated into 350 ml glass jar containing MS (Murashige and using Clorox  solution was extremely efficient for Skoog, 1962) medium supplemented with 30.0 g/l sucrose and 2.0 establishing aseptic buds of other Zingiberaceae species. g/l gelrite for 30 days. The pH of the medium was adjusted to 5.7 prior to autoclaving at 121°C for 21 min. The cultures were Similar sterilization process was also used to establish maintained in a culture room with 16/8 h photoperiod (light/dark) at the aseptic buds of Cymbopogon nardus (Chan et al., 25 ± 2°C. After 30 days, the aseptic buds of this species were 2005). Once the contamination free cultures of the shoot transferred onto MS medium supplemented with 3.0 mg/l BAP and buds were established, they were easily maintained by 0.5 mg/l NAA, the optimum medium was formulated for other sub culturing on fresh medium. Zingiberaceae species by Yusuf et al. (2007) for shoot multiplication for four (4) weeks (Figure 1D). The multiple shoots produced were separated and transferred into MS medium devoid of plant growth regulators for four (4) weeks and were used for all subsequent Shoots growth and multiplication experiments. The aseptic shoots of B. rotunda cultured on MS medium supplemented with different concentrations of BAP alone Induction of multiple shoots (0.0 to 5.0 mg/l) or in combinations with NAA (0.0 to 2.0 mg/l) resumed their growth, produced shoots and roots, Isolated shoot buds were inoculated onto MS (Murashige and Skoog, 1962) medium containing 30.0 g/l sucrose and 2.0 g/l gelrite simultaneously (Table 1). The simultaneous production of as solidifying agent supplemented with cytokinins (6-benzyla- shoots and roots were also reported in other Zingibe- minopurine (BAP)) ranging from 0.0, 0.5, 1.0, 2.0, 3.0 and 5.0 mg/l raceae species (Balachandran et al., 1990; Chan and and auxin (α-naphthaleneacetic acid (NAA)) ranging from 0.0, 0.5, Thong, 2004; Bharalee et al. , 2005). The growth of the 1.0 and 2.0 mg/l, for shoot multiplication. A single shoot was shoots and subsequent multiplication could not be cultured into each 350 ml glass jar and 10 experimental units were achieved in medium without growth regulators. After 4 to used for each medium combination. 6 weeks, about 90% of the shoots of B. rotunda that were

cultured on MS medium supplemented with 0.5 to 3 mg/l

Scoring of data of BAP and 0.5 mg/l NAA showed different levels of development with high micropropagation frequency All cultures were examined periodically, and the morphological (induced 4 to 5 multiple shoots, Table 1) and variable changes were recorded on the basis of visual observations and number of leaves with 10 to 15 roots hairs including were made after one to four weeks of culture for four consecutive secondary roots per explants (data not shown). The subcultures. There were 10 cultures per treatment for shoot multi- maximum number of multiple shoots (5) was obtained in plication and subculturing was carried out at an interval of four weeks. The effect of different treatments and subcultures were the medium containing 2.0 mg/l of BAP and 0.5 mg/l NAA quantified on the basis of percentage of cultures showing response four (4) weeks after culture initiation (Figure 2). However, for multiple shoots formation. the buds cultured on MS medium containing higher 1196 Afr. J. Biotechnol.

Figure 1. In vitro regeneration and plant establishment of B. rotunda . (A) Shoots sprouted from mature rhizomes of B. rotunda . Bar represents 25 mm. (B) Sprouted buds (30 to 50 mm) collected as explants. (C) Shoots after been surface sterilized under aseptic conditions with external leaves removed and trimmed down (5 to 8 mm) as explants. Bar represents 5 mm. (D) Induction of multiple shoots of B. rotunda on MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l NAA after four (4) weeks of culture. Bar represents 15 mm. (E) Formation of roots on acclimatized semi-solid MS medium devoid of plant growth regulators with 2.0% (w/v) activated charcoal after four (4) weeks of culture. Bar represents 20 mm. (F) The in vitro -derived plants germinated in soil with normal morphology. Bar represents 100 mm.

concentration of BAP (5.0 mg/l) and NAA (2.0 mg/l) multiplication rate but was BAP-dependent. Multiplication showed low level of development and low micropropaga- frequency was increased until BAP concentration tion frequencies (1 to 2 shoots per explant) and also reached 3.0 mg/l. showed abnormalities; even though it produced multiple Since there was no increase in the number of shoots shoots, the plantlets were stunted and the leaves became formed and as it would also be economically feasible, low yellowish within four (4) weeks of culture. These results concentration of BAP (2.0 mg/L) and NAA (0.5 mg/L) indicated that there was an optimal concentration of plant supplemented into MS medium formulation was chosen growth regulators required for normal shoot multiplication as the shoot multiplication medium for B. rotunda . Other of in vitro cultures of B. rotunda species. Balachandran et researchers reported higher concentration of plant growth al. (1990) also reported that higher concentration of regulators for the induction of multiple shoots formation kinetin was not suitable for Zingiber officinale . Aseptic for some of the Zingiberaceae species. Loc et al. (2005) cultures in MS supplemented with 2.0 mg/l BAP and 1.0 reported that MS medium supplemented with 20% (v/v) mg/l NAA induced more roots than shoots. Micropro- coconut water, 3 mg/l BA and 0.5 mg/l IBA, could induce pagation was quantified by the number of shoot. Well- the formation of an average of 5.6 shoots per explant for formed shoots was BAP-dependent and was inhibited by Curcuma zedoaria . Bharalee et al. (2005) found that MS NAA. The medium having NAA alone had no effect on medium supplemented with 4 mg/l BAP and 1.5 mg/L shoot multiplication or growth. The increase of NAA NAA was the best medium for shoot multiplication of C. concentration higher than 1.0 mg/l sup-pressed caesia (average 3.5 shoots per explant) and MS plus 1.0 Yusuf et al. 1197

Table 1. Effects of MS medium supplemented with BAP (0, 0.5, 1.0, 2.0, 3.0 and 5 mg/l) and NAA (0, 0.5, 1.0 and 2.0 mg/l) on multiple shoots formations of B. rotunda over a period of four (4) weeks of culture.

BAP (mg/l) NAA (mg/l) Mean no. of multiple shoots 0.0 0.0 1.0 ± 0.0 0.5 0.0 2.9 ± 0.2 1.0 0.0 3.3 ± 0.2 2.0 0.0 4.1 ± 0.3 3.0 0.0 3.9 ± 0.3 5.0 0.0 2.7 ± 0.2 0.0 0.5 1.0 ± 0.0 0.5 0.5 3.8 ± 0.1 1.0 0.5 4.2 ± 0.1 2.0 0.5 4.9 ± 0.1 3.0 0.5 4.0 ± 0.3 5.0 0.5 2.8 ± 0.2 0.0 1.0 1.0 ± 0.0 0.5 1.0 2.7 ± 0.3 1.0 1.0 2.9 ± 0.3 2.0 1.0 3.6 ± 0.4 3.0 1.0 3.2 ± 0.4 5.0 1.0 2.4 ± 0.3 0.0 2.0 1.0 ± 0.0 0.5 2.0 2.4 ± 0.3 1.0 2.0 2.5 ± 0.2 2.0 2.0 2.8 ± 0.2 3.0 2.0 2.6 ± 0.3 5.0 2.0 1.8 ± 0.2

Values represent means ±SE for 10 cultures per treatment.

Figure 2. Effects of MS medium supplemented with BAP (0, 0.5, 1.0, 2.0, 3.0 and 5 mg/l) and low concentration of NAA (0.5, mg/l) on multiple shoots formations of B. rotunda over a period of four (4) weeks of culture. Values are mean ± standard deviation for 10 cultures per treatment. 1198 Afr. J. Biotechnol.

regulator(s) could be potentially used for the in vitro germplasm conservation of B. rotunda . The effect of subculture on shoot multiplication was tested in MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l NAA for 16 weeks. The results indicated that the number of multiple shoots were low during initial subcultures, and increased to 5.0 ± 0.3 at 3rd subculture and then decreased slightly to 4.4 ± 0.5 at 4th subculture (Figure 3). In vitro plantlets of B. rotunda with well developed roots were successfully acclimatized with 85% survival of the plantlets when transplanted to the field environment. In vitro plantlets were morphologically similar to their respective mother plants (Figure 1F).

Conclusion

A supplementation of 2.0 mg/l BAP and 0.5 mg/l NAA gave the best result to stimulate a successful production Subculture of multiple shoots of B. rortunda with 90% of the inoculated explants inducing multiple shoots. Clonal propa- Figure 3. Effects of subculture on multiple shoots formations of B. gation and germplasm conservation of B. rotunda was rotunda over a period of four (4) subcultures in MS medium attractive because the propagated material was shown to supplemented with 2.0 mg/l BAP and 0.5, mg/l NAA. Values are possess high genetic stability (Rout et al., 1998). In vitro mean ±standard deviation for 10 cultures per treatment. plantlets can be an important source of disease-free planting material, ideally suited for germplasm exchange, transportation and conservation. mg/L BAP and 0.5 mg/L NAA for C. zedoaria (average 4.5 shoots per explant). Balachandran et al. (1990) reported that C. domestica C. caesia and C. aeruginosa REFERENCES could produced an average of 3.4, 2.8 and 2.7 shoots per explant respectively using MS medium supplemented Balachandran SM, Bhat SR, Chandel KPS (1990). In vitro clonal multiplication of turmeric ( Curcuma spp) and ginger ( Zingiber with 3 mg/L BAP. Nayak (2000) reported MS medium officinale Rosc ). Plant Cell Rep. 8: 521-524. supplemented with 5 mg/L BAP was most effective for Bhagyalakshmi Singh NS (1988). Meristem culture and shoot multiplication of C. aromatic producing an average micropropagation of a variety of ginger ( Zingiber officinale Rosc ) with 3.3 shoots per explants. Similar results indicating a high yield of oleoresin . J. Hortic. Sci . 63: 321-327. Bharalee R, Das A, Kalita MC (2005). In vitro clonal propagation of cytokinin and auxin effects on shoot multiplication had Curcuma ceasia Roxb. and Curcuma zedoaria Rosc. From rhizome been reported earlier in ginger (Hu & Wang, 1983; Palai bud explants. J. Plant Biochem. Biotechnol. 14: 61-63. et al., 1997). Hosoki and Sagawa (1977) reported that the Borthakur M, Bordoloi DN (1992). Micropropagation of Curcuma amada requirement of a cytokinin for high-frequency shoot Roxb. J. Spice Aromatic Crops, 1: 154-156. Chan LK, Thong WH (2004). In vitro propagation of Zingiberaceae multiplication of ginger was as high as 5.0 mg/L IBA. In species with medicinal properties. J. Plant Biotechnol. 6: 181-188. addition, Palai et al. (1997) indicated that the combination Chan L, Dewi PR, Boey PL (2005). Effect of plant growth regulators on of two cytokinins along with auxin increased the rate of regeneration of plantlets from bud cultures Cymbopogon nardus L. shoot multplication (16-fold) within four (4) weeks of and the detection of essential oils from the in vitro plantlets. J. Plant Biol. 48: 142-146. culture. The present findings suggested that a high Fahey JW, Stephenson KK (2002). Pinostrobin from honey and Thai frequency of shoot production from a single shoot ginger ( Boesenbergia pandurata ): a potent flavanoid inducer of meristem could be achieved by manipulating the plant mammalian phase 2 chemoprotective and antioxidant enzymes. J. growth regulators. Agric. Food Chem. 50(25): 7472-7476. Hosoki T, Sagawa Y (1977). Clonal propagation of ginger ( Zingiber The number of shoots generated from B. rotunda shoot officinale Rosc.) through tissue culture. Hort. Sci. 12: 451-452. explants cultured on the proliferation medium supple- Hu CY, Wang PJ (1983). Meristem, shoot tip and bud culture. In: Evan mented with 2.0 mg/l BAP and 0.5 mg/l NAA remained DA, Wang WR, Ammirato PV, Yamada Y (Eds.). Handbook of plant unchanged after either four (4) or eight (8) weeks of sub- cell culture, Macmillan Publishing Co. New York. pp. 177-277. Ikeda LR, Tanabe MJ (1989). In vitro subculture application for ginger. culturing. Thus, the in vitro plantlets could be subcultured Hort. Sci. 24: 142-143. every eight (8) weeks in terms of culture preservation. In Inden H, Asahira T (1988). Micropropagation of ginger. Acta Hortic. 230: addition, the in vitro plantlets could be maintained on 177-184. solid basic MS medium for two (2) months without any Jaipetch T, Kanghae S, Pancharoen O, Patrock VA, Reutrakul V, Tuntiwachwuttikul P, White AH (1982). Constituents of Boesenbergia subculturing been required and the plants remain healthy. pandurata . Aust. J. Chem. 35: 351-361. Hence MS medium without the addition of plant growth Larsen K (1996). A Preliminary checklist of the Zingiberaceae of Yusuf et al. 1199

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